This is an application for a Ruth L. Kirschstein NRSA for Individual Predoctoral Fellowship to Promote Diversity in Health-Related Research (Parent F31- Diversity) award for Antentor Othrell Hinton, Jr., a graduate student at Baylor College of Medicine. Hypertension (HTN) is the leading cause of cardiovascular disease worldwide and the number one killer in women. Interventions that treat and prevent HTN are urgently needed. Psychological stress contributes to development of HTN in humans, and these effects are mediated at least partly by neurons in the amygdala. In humans, mice and rats, neural activities in the medial amygdala (MeA) are positively associated with levels of blood pressure (BP) during stress and estrogens attenuate stress-induced c-fos expression in the MeA via estrogen receptor-? (ER?), yet the mechanisms remain unknown. Estrogen replacement therapy (ERT) lowers BP in postmenopausal women, but it also increases the risk of stroke and thromboembolism. It is the premise of this proposal that understanding specific pathways underlying the anti-hypertensive effects of estrogens may lead to novel and safer therapies for HTN. Here we hypothesize that ER?-initiated signaling pathways in MeA neurons prevent stress-induced HTN. In Aim 1, we hypothesized that ER? in the MeA mediates estrogenic effects to prevent stress-induced HTN. We generated sim1-ER?-KO mice, in which ER? is deleted mostly from MeA SIM1 neurons. Although estrogen replacement prevented stress-induced HTN in wild type ovariectomized (OVX) females, these effects were blunted in sim1-ER?-KO mice. To confirm these findings we will stereotaxically inject AAV-Cre (vs. AAV-GFP as a control) into the MeA of lox-ER? female mice. In Aim2, we found that central administration of a selective ER? agonist, propyl pyrazole triol (PPT), in female mice increased interactions of ER? and p85a (a regulatory PI3K subunit), and increased phosphorylation of AKT (a PI3K-mediated event) in the amygdala. This led us to hypothesize that PI3K in the MeA mediates estrogenic effects to prevent stress-induced HTN. To confirm these findings, we will stereotaxically inject AAV-Cre (vs. AAV-GFP as a control) into the MeA of lox-p110a female mice. In Aim 3, preliminary data suggest that central administration of PPT stimulates nitric oxide (NO) production in the amygdala via nitrate/nitrite assay. We will test if deletion of ER? i nNOS neurons (nNOS-ER?-KO mice) will blunt anti-hypertensive effects of E2 during stress and, rescue sim1-ER?-KO female mice using a NO donor. This proposal will teach us about the role of estrogens/ER? and MeA regulation in stress-induced HTN. Our studies will prove the concept that selective activation of MeA ER? can decrease BP, and therefore may identify a novel drug target for the treatment of HTN.